Cellulose-containing plant cell walls provide an abundant and renewable source of glucose, pentoses, and other small carbon compounds, many of which have significant commercial value. For example, glucose is particularly valuable as a feedstock for yeast in the production of bioethanol. Other commercially valuable byproducts of enzymatic conversion of cellulosic materials may be used in the manufacture of chemical products such as plastics, solvents, chemical intermediates, phenolics, adhesives, furfural, fatty acids, acetic acid, carbon black, paints, dyes, pigments, inks, and detergents; in the production of power; and in food and feed products. Accordingly, there has been substantial interest in developing improved techniques for microbial enzymatic processing of cellulosic materials.
Microbial cellulases represent an enormous range of proteins with widely varying specificities, cleavage patterns, and operating parameters. Among the cellulose-degrading enzymes, there are endo-acting cellulases that cleave at internal sites on the cellulose chain, exo-acting cellulases that cleave fragments from the ends of the cellulose chain, and β-glucosidases that hydrolyze soluble fragments to glucose. The diversity of cellulases is demonstrated by their presence in seven glycoside hydrolase families (Families 1, 5, 6, 7, 9, 10, and 48). Generally, cellulase-degrading enzymes produced by aerobic bacteria are soluble, while those produced by anaerobic bacteria are bound in large, multicomponent extracellular enzyme complexes called cellulosomes.
Thermophilic, cellulase-producing microbes have been isolated and identified. In particular, known cellulase-producing thermophiles capable of growing at 70° C. or higher include both aerobes (e.g., Caldibacillus cellovorans, Rhodothermus marinus and Acidothermus cellulolyticus) and anaerobes (e.g., Anaerocellum thermophilum, Caldicellulosiruptor saccharolyticus, Clostridium thermocellum, Fervidobacterium islandicum, Spirochaetta thermophila, Thermotoga maritime and Pyrocccus furiosus).
Despite the fact that several thermophilic microorganisms are known to produce cellulases, there remains no source of thermostable cellulase suitable for commercial use for most applications, including bioethanol production. The current commercially available products are mixtures of fungal cellulases that have effective temperature ranges of from 20° C. to 50° C. Much of the research on cellulases has focused on fungal cellulase systems, particularly the cellulytic system of Trichoderma reesei. This multi-component enzyme system has many benefits, including the ability to produce high yields of glucose from acid-treated cellulose. However, the cellulase enzymes from this organism are not stable for extended periods of time at high temperatures (greater than 60° C.). Thus, they must be used at temperatures below 40° C.
Some success has been reported in improving the thermostability of cellulase compositions by either site-directed mutagenesis or by cloning more thermostable endoglucanases into T. reesei. However, the improved T. reesei enzyme products remain unsuitable for use in starch liquefaction, and it is unlikely that the thermostability of all the components in the compositions could ever be increased sufficiently for the product to work under those conditions. An additional problem with T. reesei-derived enzyme products is that the cellulosic feedstock requires extensive pretreatment before being digested with the enzymes. To obtain adequate conversion, the cellulose must be pretreated with acid, high temperature steam, ammonia, or other extreme processing conditions to break down the crystal structure of the cellulose. While these pretreatments may be acceptable for use with cellulosic materials, these pretreatments are not practical within the processes currently used in the production of bioethanol, among other commercial applications.